Promiscuous Oxidosqualene Cyclases from Neoalsomitra integrifoliola Catalyzing the Formation of Tetracyclic, Pentacyclic, and Heterocyclic Triterpenes.

Six oxidosqualene cyclases (NiOSC1-NiOSC6) from Neoalsomitra integrifoliola were characterized for the biosynthesis of diverse triterpene scaffolds, including tetracyclic and pentacyclic triterpenes from the 2,3-oxidosqualene (1) and oxacyclic triterpenes from the 2,3:22,23-dioxidosqualene (2). NiOSC1 showed high efficiency in the production of naturally rare (20R)-epimers of oxacyclic triterpenes. Mutagenesis results revealed that the NiOSC1-F731G mutant significantly increased the yields of (20R)-epimers compared to the wild type. Homology modeling and molecular docking elucidated the origin of the (20R)-configuration in the epoxide addition step.

Development of a stable genetic transformation system in Erigeron breviscapus: a case study with EbYUC2 in relation to leaf number and flowering time.

MAIN CONCLUSION: A stable genetic transformation system for Erigeron breviscapus was developed. We cloned the EbYUC2 gene and genetically transformed it into Arabidopsis thaliana and E. breviscapus. The leaf number, YUC2 gene expression, and the endogenous auxin content in transgenic plants were significantly increased. Erigeron breviscapus is a prescription drug for the clinical treatment of cardiovascular and cerebrovascular diseases. The rosette leaves have the highest content of the major active compound scutellarin and are an important component in the yield of E. breviscapus. However, little is known about the genes related to the leaf number and flowering time of E. breviscapus. In our previous study, we identified three candidate genes related to the leaf number and flowering of E. breviscapus by combining resequencing data and genome-wide association study (GWAS). However, their specific functions remain to be characterized. In this study, we cloned and transformed the previously identified full-length EbYUC2 gene into Arabidopsis thaliana, developed the first stable genetic transformation system for E. breviscapus, and obtained the transgenic plants overexpressing EbYUC2. Compared with wild-type plants, the transgenic plants showed a significant increase in the number of leaves, which was correlated with the increased expression of EbYUC2. Consistently, the endogenous auxin content, particularly indole-3-acetic acid, in transgenic plants was also significantly increased. These results suggest that EbYUC2 may control the leaf number by regulating auxin biosynthesis, thereby laying a foundation for revealing the molecular mechanism governing the leaf number and flowering time of E. breviscapus.

Functional characterization of genes related to triterpene and flavonoid biosynthesis in Cyclocarya paliurus.

MAIN CONCLUSION: The oxidosqualene cyclases (OSCs) generating triterpenoid skeletons in Cyclocarya paliurus were identified for the first time, and two uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyzing the glycosylation of flavonoids were characterized. Cyclocarya paliurus, a native rare dicotyledonous plant in China, contains an abundance of triterpenoid saponins and flavonoid glycosides that exhibit valuable pharmaceutical effects in preventing hypertension, hyperlipidemia, and diabetes. However, the molecular mechanism explaining the biosynthesis of triterpenoid saponin and flavonoid glycoside in C. paliurus remains unclear. In this study, the triterpene content in different tissues and the expression pattern of genes encoding the key enzymes associated with triterpenoid saponin and flavonoid glycoside biosynthesis were studied using transcriptome and metabolome analysis. The eight upstream oxidosqualene cyclases (OSCs) involved in triterpenoid saponin biosynthesis were functionally characterized, among them CpalOSC6 catalyzed 2,3;22,23-dioxidosqualene to form 3-epicabraleadiol; CpalOSC8 cyclized 2,3-oxidosqualene to generate dammarenediol-II; CpalOSC2 and CpalOSC3 produced ß-amyrin and CpalOSC4 produced cycloartenol, while CpalOSC2-CpalOSC5, CpalOSC7, and CpalOSC8 all produced lanosterol. However, no catalytic product was detected for CpalOSC1. Moreover, two downstream flavonoid uridine diphosphate (UDP)-glycosyltransferases (UGTs) (CpalUGT015 and CpalUGT100) that catalyze the last step of flavonoid glycoside biosynthesis were functionally elucidated. These results uncovered the key genes involved in the biosynthesis of triterpenoid saponins and flavonoid glycosides in C. paliurus that could be applied to produce flavonoid glycosides and key triterpenoid saponins in the future via a synthetic strategy.

Reconstruction of engineered yeast factory for high yield production of ginsenosides Rg3 and Rd.

Panax notoginseng is one of the most valuable traditional Chinese herbs. The main active ingredients, dammarane-type ginsenosides, show multiple pharmacological activities. Recently, the key UDP-dependent glycosyltransferases (UGTs) involved in the biosynthesis of common ginsenosides have been widely studied. However, only a few UGTs that catalyze ginsenoside formation have been reported. This study further investigated the new catalytic function of 10 characterized UGTs from the public database. PnUGT31(PnUGT94B2) and PnUGT53 (PnUGT71B8)exhibited promiscuous sugar-donor specificity of UDP-glucose and UDP-xylose, which could catalyze the glycosylation of C20-OH sites and elongation of the sugar chain at the C3 and/or C20 sites. We further analyzed the expression patterns in P. notoginseng and predicted the catalytic mechanisms of PnUGT31 and PnUGT53 using molecular docking simulations. Moreover, different gene modules were built to increase the yield of ginsenosides in engineered yeast. The metabolic flow of the proginsenediol (PPD) synthetic pathway was enhanced by LPPDS gene modules based on the engineered strain. The resulting yeast was constructed to produce 1.72 g/L PPD in a shaking flask, but cell growth was significantly inhibited. EGH and LKG gene modules were constructed to achieve high-level production of dammarane-type ginsenosides. The production of G-Rg3 controlled by LKG modules increased 3.84 times (254.07 mg/ L), whereas the G-Rd titer reached 56.68 mg/L after 96 h in shaking flask culture under the control of all modules, both of which yielded the highest values for known microbes.

Site-directed mutagenesis identified the key active site residues of 2,3-oxidosqualene cyclase HcOSC6 responsible for cucurbitacins biosynthesis in Hemsleya chinensis.

Hemsleya chinensis is a Chinese traditional medicinal plant, containing cucurbitacin IIa (CuIIa) and cucurbitacin IIb (CuIIb), both of which have a wide range of pharmacological effects, including antiallergic, anti-inflammatory, and anticancer properties. However, few studies have been explored on the key enzymes that are involved in cucurbitacins biosynthesis in H. chinensis. Oxidosqualene cyclase (OSC) is a vital enzyme for cyclizing 2,3-oxidosqualene and its analogues. Here, a gene encoding the oxidosqualene cyclase of H. chinensis (HcOSC6), catalyzing to produce cucurbitadienol, was used as a template of mutagenesis. With the assistance of AlphaFold2 and molecular docking, we have proposed for the first time to our knowledge the 3D structure of HcOSC6 and its binding features to 2,3-oxidosqualene. Mutagenesis experiments on HcOSC6 generated seventeen different single-point mutants, showing that single-residue changes could affect its activity. Three key amino acid residues of HcOSC6, E246, M261 and D490, were identified as a prominent role in controlling cyclization ability. Our findings not only comprehensively characterize three key residues that are potentially useful for producing cucurbitacins, but also provide insights into the significant role they could play in metabolic engineering.

Biosynthetic pathway of prescription bergenin from Bergenia purpurascens and Ardisia japonica.

Bergenin is a typical carbon glycoside and the primary active ingredient in antitussive drugs widely prescribed for central cough inhibition in China. The bergenin extraction industry relies on the medicinal plant species Bergenia purpurascens and Ardisia japonica as their resources. However, the bergenin biosynthetic pathway in plants remains elusive. In this study, we functionally characterized a shikimate dehydrogenase (SDH), two O-methyltransferases (OMTs), and a C-glycosyltransferase (CGT) involved in bergenin synthesis through bioinformatics analysis, heterologous expression, and enzymatic characterization. We found that BpSDH2 catalyzes the two-step dehydrogenation process of shikimic acid to form gallic acid (GA). BpOMT1 and AjOMT1 facilitate the methylation reaction at the 4-OH position of GA, resulting in the formation of 4-O-methyl gallic acid (4-O-Me-GA). AjCGT1 transfers a glucose moiety to C-2 to generate 2-Glucosyl-4-O-methyl gallic acid (2-Glucosyl-4-O-Me-GA). Bergenin production ultimately occurs in acidic conditions or via dehydration catalyzed by plant dehydratases following a ring-closure reaction. This study for the first time uncovered the biosynthetic pathway of bergenin, paving the way to rational production of bergenin in cell factories via synthetic biology strategies.

RNA-seq analysis reveals key genes associated with seed germination of Fritillaria taipaiensis P.Y.Li by cold stratification.

Seed dormancy is an adaptive strategy for environmental evolution. However, the molecular mechanism of the breaking of seed dormancy at cold temperatures is still unclear, and the genetic regulation of germination initiated by exposure to cold temperature requires further investigation. In the initial phase of the current study, the seed coat characteristics and embryo development of Fritillaria taipaiensis P.Y.Li at different temperatures (0°C, 4°C, 10°C & 25°C) was recorded. The results obtained demonstrated that embryo elongation and the dormancy-breaking was most significantly affected at 4°C. Subsequently, transcriptome analyses of seeds in different states of dormancy, at two stratification temperatures (4°C and 25°C) was performed, combined with weighted gene coexpression network analysis (WGCNA) and metabolomics, to explore the transcriptional regulation of seed germination in F. taipaiensis at the two selected stratification temperatures. The results showed that stratification at the colder temperature (4°C) induced an up-regulation of gene expression involved in gibberellic acid (GA) and auxin biosynthesis and the down-regulation of genes related to the abscisic acid (ABA) biosynthetic pathway. Thereby promoting embryo development and the stimulation of seed germination. Collectively, these data constitute a significant advance in our understanding of the role of cold temperatures in the regulation of seed germination in F. taipaiensis and also provide valuable transcriptomic data for seed dormancy for other non-model plant species.

PanaxGDB: A Comprehensive Platform for Panax.

The genus Panax is a valuable natural medicinal source used worldwide that contains high levels of triterpenoid saponins with extensive pharmacological activities. In past decades, molecular biotechnology and breeding techniques have been respectively used to generate omics data and information on cultivars primarily from Panax ginseng (ginseng), Panax quinquefolium (American ginseng), and Panax notoginseng (Sanqi) to biosynthesize valuable saponins, improve product quality, and conduct cost-controlled cultivation. Although much data have been produced, there are concerns that redundant data might be generated and that relatively scattered data might be overlooked. Therefore, many scientists desire a reliable, comprehensive omics database of the Panax genus that could save time and promote integrated analysis. Therefore, to provide all-inclusive, reliable, and valuable information on the Panax genus, PanaxGDB, an open comprehensive database that integrates data on omics and information on varieties, was established. The database contains information on nearly 600 compounds from 12 Panax species, draft genomic sequences with annotations and gene expression levels, single nucleotide polymorphisms, genome-wide association analysis based on agronomic traits, globally collected germplasm information, summaries, omics data of the Panax genus, and online versatile analytic tools. The Panax genus database will be updated when new data are released to continue serving as a central portal to boost research on the biology and functions of Panax. PanaxGDB is available at: http://panaxGDB.ynau.edu.cn.

[Identification and expression profiling of R2R3-MYB transcription factors in Erigeron breviscapus].

R2 R3-MYB transcription factors are ubiquitous in plants, playing a role in the regulation of plant growth, development, and secondary metabolism. In this paper, the R2 R3-MYB transcription factors were identified by bioinformatics analysis of the genomic data of Erigeron breviscapus, and their gene sequences, structures, physical and chemical properties were analyzed. The functions of R2 R3-MYB transcription factors were predicted by cluster analysis. Meanwhile, the expression patterns of R2 R3-MYB transcription factors in response to hormone treatments were analyzed. A total of 108 R2 R3-MYB transcription factors, named EbMYB1-EbMYB108, were identified from the genome of E. breviscapus. Most of the R2 R3-MYB genes carried 2-4 exons. The phylogenetic tree of MYBs in E. breviscapus and Arabidopsis thaliala was constructed, which classified 234 MYBs into 30 subfamilies. The MYBs in the five MYB subfamilies of A.thaliala were clustered into independent clades, and those in E. breviscapus were clustered into four clades. The transcriptome data showed that MYB genes were differentially expressed in different tissues of E. breviscapus and in response to the treatments with exogenous hormones such as ABA, SA, and GA for different time. The transcription of 13 R2 R3-MYB genes did not change significantly, and the expression patterns of some genes were up-regulated or down-regulated with the extension of hormone treatment time. This study provides a theoretical basis for revealing the mechanisms of R2 R3-MYB transcription factors in regulating the growth and development, stress(hormone) response, and active ingredient accumulation in E. breviscapus.

Identification of two UDP-glycosyltransferases involved in the main oleanane-type ginsenosides in Panax japonicus var. major.

MAIN CONCLUSION: Two UDP-glycosyltransferases from Panax japonicus var. major were identified, and the biosynthetic pathways of three oleanane-type ginsenosides (chikusetsusaponin IVa, ginsenoside Ro, zingibroside R1) were elucidated. Chikusetsusaponin IVa and ginsenoside Ro are primary active components formed by stepwise glycosylation of oleanolic acid in five medicinal plants of the genus Panax. However, the key UDP-glycosyltransferases (UGTs) in the biosynthetic pathway of chikusetsusaponin IVa and ginsenoside Ro are still unclear. In this study, two UGTs (PjmUGT1 and PjmUGT2) from Panax japonicus var. major involved in the biosynthesis of chikusetsusaponin IVa and ginsenoside Ro were identified based on bioinformatics analysis, heterologous expression and enzyme assays. The results show that PjmUGT1 can transfer a glucose moiety to the C-28 carboxyl groups of oleanolic acid 3-O-ß-D-glucuronide and zingibroside R1 to form chikusetsusaponin IVa and ginsenoside Ro, respectively. Meanwhile, PjmUGT2 can transfer a glucose moiety to oleanolic acid 3-O-ß-D-glucuronide and chikusetsusaponin IVa to form zingibroside R1 and ginsenoside Ro. This work uncovered the biosynthetic mechanism of chikusetsusaponin IVa and ginsenoside Ro, providing the rational production of valuable saponins through synthetic biology strategy.

Comparative transcriptome and metabolome analyses provide new insights into the molecular mechanisms underlying taproot thickening in Panax notoginseng.

BACKGROUND: Taproot thickening is a complex biological process that is dependent on the coordinated expression of genes controlled by both environmental and developmental factors. Panax notoginseng is an important Chinese medicinal herb that is characterized by an enlarged taproot as the main organ of saponin accumulation. However, the molecular mechanisms of taproot enlargement are poorly understood. RESULTS: A total of 29,957 differentially expressed genes (DEGs) were identified during the thickening process in the taproots of P. notoginseng. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment revealed that DEGs associated with "plant hormone signal transduction," "starch and sucrose metabolism," and "phenylpropanoid biosynthesis" were predominantly enriched. Further analysis identified some critical genes (e.g., RNase-like major storage protein, DA1-related protein, and Starch branching enzyme I) and metabolites (e.g., sucrose, glucose, fructose, malate, and arginine) that potentially control taproot thickening. Several aspects including hormone crosstalk, transcriptional regulation, homeostatic regulation between sugar and starch, and cell wall metabolism, were identified as important for the thickening process in the taproot of P. notoginseng. CONCLUSION: The results provide a molecular regulatory network of taproot thickening in P. notoginseng and facilitate the further characterization of the genes responsible for taproot formation in root medicinal plants or crops.

Molecular cloning and expression analysis of SRLK1 gene in self-incompatible Asteraceae species Erigeron breviscapus.

Based on the transcriptome data, using RACE techniques, we cloned the full-length EbSRLK1 gene in a medicinal, self-incompatible Asteraceae species, Erigeron breviscapus. Bioinformatics approaches were used to analyze the DNA and protein sequences, physical and chemical properties, and domains of the encoded protein. The full-length EbSRLK1 cDNA is 2891 base pairs (bp) with an open reading frame (ORF) of 2634 bp, which encodes the EbSRLK1 protein with 878 amino acids and an estimated molecular weight of 98.13 kD. The EbSRLK1 protein has the characteristic domain structure of S-locus receptor-like protein kinases, which contains one transmembrane domain but lacks the signal peptide. Quantitative real-time PCR (qRT-PCR) analysis showed that the EbSRLK1 gene is lowly expressed in roots, stems and leaves, but highly expressed in flowers, especially in flowers one day prior to opening. Western blot analysis showed that the EbSRLK1 protein is expressed in stems, leaves, and flowers, but is almost undetectable in roots. The EbSRLK1 protein expression is induced in self-pollinated but not in cross-pollinated E. breviscapus flowers. Cloning and expression analysis of EbSRLK1 lay a solid foundation for elucidating the role of EbSRLK1 in regulating self-incompatibility in E. breviscapus.

Illumina-based transcriptomic analysis on recalcitrant seeds of Panax notoginseng for the dormancy release during the after-ripening process.

Panax notoginseng (Burk) F.H. Chen is an economically and medicinally important plant of the family Araliacease, with seed dormancy being a key factor limiting the extended cultivation of P. notoginseng. The seeds belong to the morphophysiological dormancy (MPD) group, and it has also been described as the recalcitrant seed. To date, the molecular mechanism of dormancy release in the recalcitrant seed of P. notoginseng is unknown. In the present study, the transcript profiles of seeds from different after-ripening stages (0, 20, 40 and 60 days) were investigated using Illumina Hiseq 2500 technology. 91 979 946 clean reads were generated, and 81 575 unigenes were annotated in at least one database. In addition, the differentially expressed genes (DEGs) were identified by the pairwise comparisons. We screened out 2483 DEGs by the three key groups of 20 days vs 0 d, 40 d vs 0 d and 60 d vs 0 d. The DEGs were analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway annotation. Meanwhile, we obtained 78 DEGs related to seeds dormancy release at different after-ripening stages of P. notoginseng, of which 15 DEGs were associated with abscisic acid and gibberellin. 26 DEGs that encode late embryogenesis abundant protein and antioxidant enzyme were correlated with desiccation tolerance in seeds. In summary, the results obtained here showed that PECTINESTERASE-2-LIKE, GA-INSENSITIVE, ENT-KAURENE SYNTHASE, PROTEIN PHOSPHATASE 2C, GIBBERELLIN 2-BETA-DIOXYGENASE, SUPEROXIDE DISMUTASE, L-ASCORBATE PEROXIDASE, CATALASE, LATE EMBRYOGENESIS ABUNDANT PROTEIN DC3 and DEHYDRIN 9 were potentially involved in dormancy release and desiccation sensitivity of P. notoginseng seeds. The data might provide a basis for researches on MPD.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides.

MAIN CONCLUSION: Oleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-ß-glucuronide. Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-ß-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-ß-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

Unigene-based RNA-seq provides insights on drought stress responses in Marsdenia tenacissima.

Marsdenia tenacissima is a well-known anti-cancer medicinal plant used in traditional Chinese medicine, which often grows on the karst landform and the water conservation capacity of land is very poorly and drought occurrences frequently. We found M. tenacissima has strong drought resistance because of continuousdrought16 d, the leaves of M. tenacissima were fully curly and dying. But the leaves were fully almost recovering after re-watering 24h. The activity of SOD and POD were almost doubled under drought stress. The content of osmotic regulating substance proline and soluble sugar were three times than control group. But after re-watering, these indexes were declined rapidly. Three cDNA libraries of control, drought stress, and re-watering treatments were constructed. There were 43,129,228, 47,116,844, and 42,815,454 clean reads with Q20 values of 98.06, 98.04, and 97.88respectively.SRA accession number of raw data was PRJNA498187 on NCBI. A total of 8672, 6043, and 6537 differentially expressed genes (DEGs) were identified in control vs drought stress, control vs re-watering, and drought stress vs re-watering, respectively. In addition, 1039, 1016, and 980 transcription factors (TFs) were identified, respectively. Among them, 363, 267, and 299 TFs were identified as DEGs in drought stress, re-watering, and drought stress and re-watering, respectively. These differentially expressed TFs mainly belonged to the bHLH, bZIP, C2H2, ERF, MYB, MYB-related, and NAC families. A comparative analysis found that 1174 genes were up-regulated and 2344 were down-regulated under drought stress and this pattern was the opposite to that found after re-watering. Among the up-regulated genes, 64 genes were homologous to known functional genes that directly protect plants against drought stress. Furthermore, 44 protein kinases and 38 TFs with opposite expression patterns under drought stress and re-watering were identified, which are possibly candidate regulators for drought stress resistance in M. tenacissima. Our study is the first to characterize the M. tenacissima transcriptome in response to drought stress, and will serve as a useful resource for future studies on the functions of candidate protein kinases and TFs involved in M. tenacissima drought stress resistance.

De novo Transcriptome Characterization of Rhodomyrtus tomentosa Leaves and Identification of Genes Involved in α/ß-Pinene and ß-Caryophyllene Biosynthesis.

Plant-derived terpenes are effective in treating chronic dysentery, rheumatism, hepatitis, and hyperlipemia. Thus, understanding the molecular basis of terpene biosynthesis in some terpene-abundant Chinese medicinal plants is of great importance. Abundant in mono- and sesqui-terpenes, Rhodomyrtus tomentosa (Ait.) Hassk, an evergreen shrub belonging to the family Myrtaceae, is widely used as a traditional Chinese medicine. In this study, (+)-α-pinene and ß-caryophyllene were detected to be the two major components in the leaves of R. tomentosa, in which (+)-α-pinene is higher in the young leaves than in the mature leaves, whereas the distribution of ß-caryophyllene is opposite. Genome-wide transcriptome analysis of leaves identified 138 unigenes potentially involved in terpenoid biosynthesis. By integrating known biosynthetic pathways for terpenoids, 7 candidate genes encoding terpene synthase (RtTPS1-7) that potentially catalyze the last step in pinene and caryophyllene biosynthesis were further characterized. Sequence alignment analysis showed that RtTPS1, RtTPS3 and RtTPS4 do not contain typical N-terminal transit peptides (62-64aa), thus probably producing multiple isomers and enantiomers by terpenoid isomerization. Further enzyme activity in vitro confirmed that RtTPS1-4 mainly produce (+)-α-pinene and (+)-ß-pinene, as well as small amounts of (-)-α-pinene and (-)-ß-pinene with GPP, while RtTPS1 and RtTPS3 are also active with FPP, producing ß-caryophyllene, along with a smaller amount of α-humulene. Our results deepen the understanding of molecular mechanisms of terpenes biosynthesis in Myrtaceae.

Identification and Characterization of Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in Coptis Species.

The dried rhizomes of Coptis chinensis have been extensively used in heat clearing, dampness drying, fire draining, and detoxification by virtue of their major bioactive components, benzylisoquinoline alkaloids (BIAs). However, C. teeta and C. chinensis are occasionally interchanged, and current understanding of the molecular basis of BIA biosynthesis in these two species is limited. Here, berberine, coptisine, jatrorrhizine, and palmatine were detected in two species, and showed the highest contents in the roots, while epiberberine were found only in C. chinensis. Comprehensive transcriptome analysis of the roots and leaves of C. teeta and C. chinensis, respectively, identified 53 and 52 unigenes encoding enzymes potentially involved in BIA biosynthesis. By integrating probable biosynthetic pathways for BIAs, the jatrorrhizine biosynthesis ill-informed previously was further characterized. Two genes encoding norcoclaurine/norlaudanosoline 6-O-methyltransferases (Cc6OMT1 and Cc6OMT2) and one gene encoding norcoclaurine-7OMT (Ct7OMT) catalyzed enzymatically O-methylate (S)-norcoclaurine at C6 that yield (S)-coclaurine, along with a smaller amount of O-methylation occurred at C7, thereby forming its isomer (isococlaurine). In addition, scoulerine 9-OMT (CtSOMT) was determined to show strict substrate specificity, targeting (S)-scoulerine to yield (S)-tetrahydrocolumbamine. Taken together, the integration of the transcriptome and enzyme activity assays further provides new insight into molecular mechanisms underlying BIA biosynthesis in plants and identifies candidate genes for the study of synthetic biology in microorganisms.

[Effects of light intensity on photosynthetic capacity and light energy allocation in Panax notoginseng.] / å ‰å¼ºå¯¹ä¸‰ä¸ƒå ‰åˆèƒ½åŠ›åŠèƒ½é‡åˆ†é çš„å½±å“.

To explore the effects of light intensity on photosynthetic characteristics and light adaptation of the shade-demanding species Panax notoginseng, the responses of photosynthesis to photosynthetic photon flux density, CO2 and sunflecks in the two-year-old Panax notoginseng were investigated under different levels of light intensity (29.8%, 9.6%, 5.0%, 1.4% and 0.2% of full sunlight). Meanwhile, chlorophyll a fluorescence parameter and light energy partitioning were also recorded and calculated in the responsive process. P. notoginseng grown under 29.8% full sunlight (FL) had a lower apparent quantum yield (AQY), potential photochemical quantum yield (Fv/Fm) and potential photochemical activity (Fv/Fo), however, it had a higher maximum net photosynthetic rate (Pn max), maximum electron transport rate (Jmax), F/Fm', electron transport rate (ETR), photochemical quenching (qP) and the proportion of light energy allocated to photochemistry dissipation (ΦPS2), but the non-photochemical quenching (NPQ) was not the highest. There were no significant differences in Pn max, light compensation point (LCP), light saturation point (LSP), dark respiration rate (Rd) among 9.6%FL and 5.0% FL treatments, but these treatments had relatively higher values of NPQ, AQY, carboxylation efficiency (CE), maximum carboxylation rate (Vc max), Fv/Fm and Fv/Fo. In addition, the Pn max, CE, Vc max, Jmax, ETR,F/Fm', qP, NPQ and ΦPSII decreased with the decrease in light intensity from 5.0%FL to 0.2%FL, and the proportion of light energy allocated to fluorescent dissipation (Φf,d) were increased. Under 500 µmol·m-2·s-1 light-flecks inducting, the ΦPSⅡof P. notoginseng increased slowly with the extension of time except for the treatment of less than 5.0%FL, and under the circumstance of 1.4%FL and 0.2%FL, ΦPSⅡreached significantly a perfect result, moreover, Φf,dincreased rapidly. These results suggested that the enhancement in photosynthetic electron transport to use the light energy and the moderate photoinhibition of PS2 might avoid the irreversible oxidative damage of photosynthetic organization in P. notoginseng under high levels of light intensity. Moderate shading was beneficial to maintain its higher non-photochemical quenching ability. However, its photosynthetic capacity depressed and the proportion of light energy allocated to non-photochemical pathway increased obviously in excessive shading, and it easily caused a light oxidative damage.

Identification of candidate genes involved in isoquinoline alkaloids biosynthesis in Dactylicapnos scandens by transcriptome analysis.

Dactylicapnos scandens (D. Don) Hutch (Papaveraceae) is a well-known traditional Chinese herb used for treatment of hypertension, inflammation, bleeding and pain for centuries. Although the major bioactive components in this herb are considered as isoquinoline alkaloids (IQAs), little is known about molecular basis of their biosynthesis. Here, we carried out transcriptomic analysis of roots, leaves and stems of D. scandens, and obtained a total of 96,741 unigenes. Based on gene expression and phylogenetic relationship, we proposed the biosynthetic pathways of isocorydine, corydine, glaucine and sinomenine, and identified 67 unigenes encoding enzymes potentially involved in biosynthesis of IQAs in D. scandens. High performance liquid chromatography analysis demonstrated that while isocorydine is the most abundant IQA in D. scandens, the last O-methylation biosynthesis step remains unclear. Further enzyme activity assay, for the first time, characterized a gene encoding O- methyltransferase (DsOMT), which catalyzes O-methylation at C7 of (S)-corytuberine to form isocorydine. We also identified candidate transcription factor genes belonging to WRKY and bHLH families that may be involved in the regulation of IQAs biosynthesis. Taken together, we first provided valuable genetic information for D. scandens, shedding light on candidate genes involved in IQA biosynthesis, which will be critical for further gene functional characterization.

De novo Sequencing and Transcriptome Analysis of Pinellia ternata Identify the Candidate Genes Involved in the Biosynthesis of Benzoic Acid and Ephedrine.

BACKGROUND: The medicinal herb, Pinellia ternata, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL) and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. RESULTS: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeq™ 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternata, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Also, 14,468 simple sequence repeats (SSRs) were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. CONCLUSION: RNA-seq data was used for the first time to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.